1. Field of the Invention
The invention relates to glycoprotein synthesis, and more particularly, to the use of a recombinant Endo D, an Endo-β-N-acetylglucosaminidase, that possesses transglycosylation activity with sugar oxazoline with limited hydrolyzing activity thereby providing for efficient glycosylation remodeling of IgG1-Fc domain.
2. Description of the Related Art
Endo-β-N-acetylglucosaminidases (ENGases) are a class of glycoside hydrolases that hydrolyze the β-1,4-glycosidic bond in the N,N′-diacetylchitobiose core of N-glycans. The deglycosylation property of ENGases has been frequently used for structural and functional studies of glycoproteins. ENGases are classified into two classes of the glycoside hydrolase (GH) families in the CAZY database, GH18 and GH85. The commonly used bacterial endo-β-N-acetylglucosaminidases, such as Endo-H from Streptomyces plicatus (1) and Endo-F1, Endo-F2, and Endo-F3 from Flavobacterium meningosepticum (2,3), belong to the GH18 family. In contrast, the GH85 family includes ENGases from both prokaryotes and eukaryotes, including Endo-M from Mucor hiemalis (4,5), Endo-A from Arthrobacter protophormiae (6), Endo-D from Streptococcus pneumoniae (7), and Endo-CE from Caenorhabditis elegans (8). In addition to the hydrolytic activity, some of the ENGases have been shown to possess transglycosylation activity, capable of transferring the released N-glycan to an alcoholic acceptor such as an N-acetylglucosamine (GlcNAc) moiety to reconstitute the natural β-1,4-glycosidic linkage. The transglycosylation activity of ENGases has attracted much attention in recent years for chemoenzymatic synthesis of oligosaccharides, glycopeptides, and glycoproteins (9, 10).
Structural and mechanistic studies on Endo-H (11), Endo-F1 (12), Endo-F3 (13), Endo-A (14, 15), and Endo-D (16) suggest that the ENGase-catalyzed N-glycan hydrolysis follows a substrate-assisted mechanism. In this mechanism, a general acid/base residue (Asp or Glu) first acts as a general acid to protonate the glycosidic oxygen. Upon activation, the 2-acetamide group of the (−1)GlcNAc in the substrate acts as a nucleophile to attack the anomeric center, resulting in the breakdown of the glycosidic bond with simultaneous formation of an oxazolinium ion intermediate. The oxazolinium intermediate then undergoes hydrolysis or transglycosylation via its reaction with a water molecule or an alcoholic acceptor activated by the general acid/base residue. These structural studies also identified another important residue, which is located at 1 or 2 amino acid residues upstream from the general acid/base catalytic residue. This key residue was shown to be an Asp residue for the GH18 ENGases (Endo-H, Endo-F1, and Endo-F3) or an Asn residue for the GH85 ENGases (Endo-A, Endo-M, and Endo-D), which was required for the proper orientation of the acetamide group to promote the oxazolinium ion formation. The essential role of this residue for hydrolysis was confirmed by the fact that mutation of this residue abolished the hydrolytic activity of ENGases (11-16).
These mechanistic and mutagenesis studies laid the basis for exploring synthetic sugar oxazolines as donor substrates for transglycosylation, which resulted in significant enhancement of the transglycosylation efficiency for glycopeptide and glycoprotein synthesis (17-22). Moreover, it was further demonstrated that novel glycosynthases could be generated by site-directed mutation at the critical Asn residue that promotes oxazolinium intermediate formation in hydrolysis (Asn-175 in Endo-M and Asn-171 in Endo-A). The resulting mutants such as EndoM-N175Q and EndoA-N171A were able to take the activated sugar oxazolines for transglycosylation with marginal or abolished hydrolytic activity on the transglycosylation product (23-29). These discoveries open a new avenue to glycoprotein synthesis and glycosylation remodeling and specifically, immunoglobulin G (IgG) antibodies.
A typical IgG antibody is composed of two light and two heavy chains that are associated with each other to form three major domains connected through a flexible hinge region: the two identical antigen-binding (Fab) regions and the constant (Fc) region. It is noted that there are heterogeneous glycosylation states of the human IgG when expressed in mammalian cell lines (e.g., CHO cell lines), and isolation of human IgG having a particular glycosylation state from this mixture is extremely difficult. Small amounts of impurities of a highly active species can dramatically interfere with the results and data interpretation. Generation of high-affinity mAbs to Fc receptors, such as the Fc γIIIa receptor, may overcome the problem of polymorphism of the Fcγ receptor variants, thus enhancing the clinical efficacy of therapeutic mAbs.
Endo-D from S. pneumoniae belongs to the glycoside hydrolase family 85 (GH85). However, in contrast to Endo-A and Endo-M that belong to the same family, Endo-D is able to hydrolyze fucosylated N-glycan core. Fairbanks and co-workers (30) first reported that Endo-D possessed transglycosylation activity, capable of using Man3GlcNAc oxazoline as donor substrate to glycosylate a GlcNAc acceptor, but the transglycosylation efficiency was very low.
In light of the above known activities of Endo D, it would be advantageous to provide a mutant Endo-D that exhibits reduced hydrolyzing activity with increased transglycosylating activity.